The reaction rate constants of enzyme catalyzed reactions are being determined under steady-state conditions by non-invasive NMR spectroscopic techniques. The 2D NMR technique permits the simultaneous determination of all of the rate constants involved in a reaction sequence, and explicitly displays them in the form of a 2D plot. In addition, the 2D NMR experiment also provides information on the relative size of the substrate pools involved in the reaction, critical information for determining reaction rates in the compartmentalized cell cytosol, but cannot detect exchanges from very small substrate pools. In contrast saturation transfer permits the monitoring of exchange processes between very small substrate pools, i.e. enzyme substrate complexes, and large pools. Four in vitro enzyme systems have been studied; phosphoglucose isomerase, carbonic anhydrase, adenylate kinase and creatine kinase. In all three cases, unique information of both the mechanism and flux characteristics of the reactions have been obtained. Due to the non-invasive nature of these techniques, they were applicable to the determination of enzyme reaction rates within in vivo tissues. We have obtained this rate data from the in vivo brain and leg of anesthetized rats. The creatine kinase catalyzed exchange between CrP and ATP was observed in both tissues, while the ATP hydrolysis-resynthesis rate was too slow to detect in these intact tissues.